TROUBLESHOOTING

Cell thawing/counting

When cryovials are delivered in dry ice, the vials in the shipping container must be covered with dry ice.

Upon receipt, cryovials must be stored in liquid (-196°C) or vapour phase (-160°C) nitrogen. Keep the cells in liquid or vapour phase nitrogen until immediately prior to thawing. Do not store the cells at -80°C more than 2 days (including shipment time on dry ice). Do not repeatedly remove the vials from the storage tank (when looking for other samples stored in the same rack), as doing so will mean they are exposed to room temperatures (even for a minute) which will compromise the quality of the cells once they are thawed. Ideally, the HepaRG cell vials should be stored separately from other products.

Cells were thawed incorrectly

Follow the procedure for thawing cells exactly as recommended by BPI. Make sure that you thaw the frozen cells quickly, and dilute them using pre-warmed medium.

Thawing medium is not correct or is the incorrect temperature.

Use the medium recommended by BPI. Check that the waterbath is at 37°C using a reliable thermometer (do not rely solely on the display on the waterbath). Make sure the medium is pre-warmed and remains in the waterbath until the cells are thawed. Transfer the vial and the warmed thawing medium into the flow hood at the same time.

Cells were not handled gently

Freezing and thawing procedures are stressful for cells. Do not vortex them, or centrifuge the cells at high speed.

Counting difficulty

Perform a repeat count with a new sample. Avoid vortexing the cells to mix them.

Shipment: temperature not maintained below -70°C

Normally, cryopreserved cells are shipped in a vapour phase nitrogen cryoshipper, which maintains the temperature at ~-160°C for at least 7 days. However, when cryovials are delivered in dry ice, the vials in the shipping container must be covered with dry ice. We pack the vials to try to ensure this does not happen but if the shipment takes 2 days, there is less dry ice in the box. It is important to note whether the vials are immersed in the dry ice when they arrive.

Unexpected cell number

Improper thawing technique

Review BPI thawing and counting protocols.

Thawing medium is not correct

Use the medium recommended by BPI.

Centrifugation speed is not correct.

Follow protocol for centrifugation exactly as recommended by BPI, and be sure the centrifuge is calibrated. If the viability is very high but the cell number is low, then the centrifugation speed is too slow - increase the speed and repeat centrifugation (this may require some optimisation). By contrast, if the viability is lower than expected and the number of cells is very high, then the centrifugation speed is too high and is pelleting dead cells as well as viable cells.

Loss of cells

After centrifugation, aspirate the supernatant while leaving a small volume on the pellet. Be careful not to aspirate the pellet.

Improper counting technique

Ensure a homogeneous cell mixture prior to counting. Due to the normal presence of aggregates in the cell suspension, we recommend counting the cells with a Nageotte hemocytometer which has a deeper chamber compared to other hemocytometers (e.g. Malassez or Neubauer hemocytometer). Such a hemocytometer will allow for visualization of all the cells; use of hemocytometers with shallow chambers can mean some cells are not counted. Count all the cells, including cells in aggregates, across four or more lines.

Review BPI thawing and counting protocols and confirm that all steps were carried out as described - deviation from the protocol may result in poorer quality and fewer cells.

Cell culture

Low attachment efficiency

Poor-quality matrix

Use only Collagen I coated plates from recognized manufacturers (e.g. those made at BPI that are quality controlled specifically for use with HepaRG™ and primary hepatocytes).

Cells not handled gently

Freezing and thawing procedures are stressful for cells causing them to be fragile. Handle the cells gently.

Sub-optimal monolayer confluency

Low Seeding density

Follow procedure for seeding the cells exactly as recommended by BPI.

Low attachment efficiency; inadequate time allowed for attachment

Low attachment efficiency may be due to:1) Poor-quality matrix. Use only Collagen I coated plates from recognized manufacturers (e.g. those made at BPI that are quality controlled specifically for use with the HepaRG™ and the hepatocytes).2) Cells not handled gently. Freezing and thawing procedures are stressful for cells and causes them to be fragile. Handle the cells gently.

Poor cell distribution in wells

Insufficient cell dispersion during plating

Disperse cells evenly by moving plate in a back-and-forth and side-to-side manner.Don’t rotate the plate as this causes the cells to gather in the centre of the well and this decreases the overall attachment of cells.

Cell morphology alteration

Media deficiencies

The use of appropriate culture media is crucial to the optimal health of cells. Please follow our recommendations; use of non-recommended materials can cause significant harm to HepaRG™ cells.Make sure the storage conditions are adhered to and do not use expired media or supplements. The pH of working medium is also important. To prevent rapid alkalinization of the medium, do not allow it to be at room temperature for more than an hour. Never add Hepes to the medium for maintaining the pH.Perform medium changes as recommended.

May be explained by:1) Low seeding density. Follow procedure for seeding the cells exactly as recommended by BPI. If the cells were not seeded at the correct density, then they will behave differently compared to what is expected. Cell-cell contact is essential to optimal performance.2) Low attachment efficiency. See comments and suggestions listed for this problem.

Toxicity

Check that none of the medium components are present at the wrong concentration.

Hole in the cell monolayer

Pipetting is too forceful during the medium changes.

During the medium changes, transfer the culture medium gently on the side of the wells.

Scratch in the cell monolayer

During the medium changes, tearing of the cell monolayer while aspirating the medium

During the medium changes, take care while aspirating the medium not to touch the cell monolayer.

Observation of threads in the culture

Formation of matrix filaments, which is normal but can appear to be a fungal contamination

The filaments are formed by the extracellular matrix normally produced by the cells.

Observation of small particules in the culture

Natural presence of particules in the serum

Particules are naturally present in the serum and do not have adverse effects on the cells.

Rapid pH shift in medium

Incorrect percentage of CO2 in the incubator

Check if your incubator is at 5% CO2.

Bacterial, yeast, or fungal contamination

Discard the culture, and carefully check culture media for contamination.Decontaminate material and equipment.

Medium

Colour change of the medium from orange to purplish

pH change on open bottle

To prevent rapid alkalinization of the medium, do not allow it to be at room temperature for more than an hour. Never add Hepes to maintain the pH.When working medium seems a bit purplish, it can be placed in a 5% CO2 incubator with the cap slightly unscrewed to re-equilibrate the pH for maximum 2 hours. The color should come back to normal. Make sure the storage conditions are adhered to and do not use expired media or supplements.

Small particles in the medium

Particles brought by the serum.

The particles observed in the medium may come from the serum. After mixing the additives with the base medium, the working medium can either be used as it is (homogenize the medium with rotating movements before use) or filtered to remove particles in a 0.2µm filter.